Anthrax vaccine formulation and uses thereof

ABSTRACT

An anthrax vaccine with alhydrogel as the adjuvant is presented in a composition formulated by mixing rPA with colloidal alhydrogel (alum) adjuvant to produce the final product, comprising 200 μg/ml rPA bound to 0.26% alhydrogel in phosphate buffered saline (PBS), said formulation containing an optimal concentration of phosphate. Methods of using the vaccines to treat or prevent infections are also presented.

This application is a National Phase of International ApplicationPCT/GB2009/051293, filed 2 Oct. 2009, which claims priority of U.S.Provisional Application 61/194,967, filed 2 Oct. 2008, the disclosure ofwhich is hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS NOTICE

The present invention was produced under government contractN01-AI-30052 with the U.S. National Institutes of Allergy and InfectiousDiseases (NIAID), which was subsequently transferred to U.S. BiomedicalAdvanced Research and Development Authority (BARDA) (and renumberedHSSO100200900103C) so that the U.S. Government may have rights in anypatent that issues.

FIELD OF THE INVENTION

The present invention relates to the field of vaccine formulations, suchas that containing anthrax protective antigen, and uses of such vaccine,whereby efficacy of such vaccine is both maintained and enhanced.

BACKGROUND OF THE INVENTION

Vaccines are commonly formulated using an adjuvant. A commonadjuvant-type is the aluminum based colloids, usually referred to asalum. More specifically these are usually aluminum hydroxide (aluminumoxyhydroxide) (also called alhydrogel) and aluminum phosphate (alsocalled adju-phos). For example, vaccines useful against organisms suchas anthrax (Bacillus anthracis) are commonly formulated with alhydrogel,which binds the anthrax antigen used in such vaccines (so called subunitvaccines). One aim of such product formulations was to maximize thebinding of rPA to the alum. Since phosphate ions were known to desorbantigen from the alhydrogel colloid, the phosphate concentration in suchformulations has been kept deliberately low (at 0.25 mM). At thisconcentration the phosphate buffer does not interfere with rPA bindingto the alhydrogel colloid. For example, with this formulation we havefound that recombinant protective antigen (rPA) binding was >98%.

Batches of this drug product formulation have been used in the Phase Iand Phase II clinical studies, which demonstrated safety andimmunogenicity in man.

With this original formulation the pH was found to be 5.9 due to theinsufficient buffering capacity of the low phosphate buffer. In order toincrease the stability of the rPA drug product and provide a morephysiological pH, it was decided that increased control of theformulation was required. The pH of the drug product formulation had tobe increased to pH 7 so that an improvement in the buffering capacity ofthe formulation would be required. For control at pH 7.0, a possiblephysiological buffer is phosphate, which has a pKa at 7.2 but thedisadvantage of using phosphate in the formulation was the inhibitoryeffect upon rPA-alum binding.

Based on pKa, the possible alternative would be histidine (pKa 6.04),however when stored as a liquid, it has the propensity to oxidize andproduce a brown coloration. Moreover, introduction of an alternativebuffer such as histidine would mean a radical change in the formulation.A new approach was therefore needed.

We subsequently discovered that we could increase phosphateconcentration to a level that both controlled pH and afforded minimaleffect on rPA:alhydrogel binding. Consequently, the phosphateconcentration was increased to approximately 4 mM; a concentration thatwas capable of maintaining the Drug Product at a pH of approximately7.0, and did not markedly affect the amount of unbound rPA, whichremained below the level of detection of the assay (<2%).

This led to the surprising result that such a phosphate concentrationalso greatly increases the bioactivity of the vaccine formulation.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an anthrax vaccinecomposition, comprising a therapeutically effective amount of an anthraxantigen in a pharmaceutically acceptable carrier and containing aphosphate salt at a concentration in the range of 2 mM to 10 mM,preferably 2.5 mM to 7.5 mM, more preferably 3 mM to 5 mM, or in therange of 3.5 mM to 4.5 mM, even more preferably in the range of 3.8 mMto 4.2 mM, or in the range of 3.9 mM to 4.1 mM and most preferably isabout 4.0 mM.

In specific embodiments, the vaccine comprises an anthrax antigen,preferably an anthrax subunit such as protective antigen, mostpreferably recombinant protective antigen (rPA). In other embodiments,the vaccine further comprises an adjuvant, preferably alum (alhydrogel).In one embodiment, the vaccine composition is in the form of alyophilized powder. In another embodiment, the vaccine composition mayalso comprise phosphate buffered saline (PBS).

In other examples of the vaccine composition or formulation of theinvention, the pH of the vaccine composition is in the range of 7.0 to7.2, preferably about 7.1.

In other examples, the anthrax vaccine formulation of the inventioncontains alhydrogel (alum) at about 0.15 to 0.35%, preferably at about0.20 to 0.30%, more preferably at about 0.24 to 0.28%, and mostpreferably wherein the alhydrogel (alum) is present at about 0.26%.

In a preferred embodiment, the vaccine of the invention is a sub-unitvaccine. In a further embodiment, such sub-unit vaccine is an anthraxvaccine of comprising rPA is present at about 200 μg/ml, whereinphosphate is present at about 4 mM, wherein alhydrogel is present atabout 0.26% by weight and wherein the pH of said vaccine is about 7.1.

The present invention further relates to a method of protecting againsta bacterial infection in a mammal, comprising administering to a mammalat risk of such infection a therapeutically-effective amount of thevaccine composition of the invention. In further embodiments thereof,the bacterial infection is an anthrax (Bacillus anthracis) infectionand/or the mammal is a human being.

The present invention also relates to a method of treating a bacterialinfection in a mammal, comprising administering to a mammal afflictedwith such infection a therapeutically-effective amount of the vaccinecomposition of the invention. In further embodiments thereof, thebacterial infection is an anthrax (Bacillus anthracis) infection and/orthe mammal is a human being.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flowchart for manufacture of a recombinant ProtectiveAntigen (rPA) drug product.

FIG. 2 shows the results of an ED50 comparison of high and low phosphatedrug products.

FIG. 3 shows the effect of phosphate on the zeta potential of alhydrogeldiluent and drug product.

FIG. 4 shows the effects of phosphate on unbound rPA.

FIG. 5 shows the effect of phosphate on adsorptive capacity andadsorptive coefficient of rPA binding to alhydrogel as determined byLangmuir analysis.

FIG. 6 shows the effect of phosphate on secondary structure of rPA in aformulated drug product.

FIG. 7 shows the effect of phosphate on tertiary structure of rPA informulated Drug Product.

FIG. 8 shows the effect of phosphate on rPA melting as determined byDSC.

FIG. 9 shows the effect of phosphate on rPA melting as determined bythermal denaturation/intrinsic fluorescence.

FIG. 10 shows epitope recognition of Drug Product formulated atdifferent phosphate concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an anthrax subunit vaccine formulationcontaining comprising a therapeutically effective amount of an anthraxantigen, such as anthrax protective antigen (rPA) in a pharmaceuticallyacceptable carrier and containing a phosphate salt at a concentration ofbetween 2 and 10 mM, preferably between 3 and 5 mM, more preferablyabout 4 mM, using alhydrogel as adjuvant. The present invention alsoprovides methods for treating or preventing bacterial infections,especially infections of anthrax in a human being, by administering atherapeutically effective amount of a vaccine of the invention.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of a bacterial infection, especially anthrax infection.The precise dosage may vary with such factors as the age, immune systemstatus, general health, and environmental circumstances of the patient,the nature and extent of the infection (or anticipated infection) andthe availability of subsequent treatment/vaccination.

Generally, vaccines are prepared as injectables, in the form of aqueoussolutions or suspensions. Vaccines in an oil base are also well knownsuch as for inhaling. Solid forms which are dissolved or suspended priorto use may also be formulated. Pharmaceutically acceptable carriers,diluents and excipients are generally added that are compatible with theactive ingredients and acceptable for pharmaceutical use.

The pharmaceutical compositions useful herein also contain apharmaceutically acceptable carrier, including any suitable diluent orexcipient, which includes any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable carriers include, but are not limited to,liquids such as water, saline, glycerol and ethanol, and the like,including carriers useful in forming sprays for nasal and otherrespiratory tract delivery or for delivery to the ophthalmic system. Athorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES(Mack Pub. Co., N.J. current edition).

Vaccine compositions may further incorporate additional substances tostabilize pH, or to function as adjuvants, wetting agents, oremulsifying agents, which can serve to improve the effectiveness of thevaccine.

Vaccines are generally formulated for parenteral administration and areinjected either subcutaneously or intramuscularly. Such vaccines canalso be formulated as suppositories or for oral administration, usingmethods known in the art, or for administration through nasal orrespiratory routes.

The amount of vaccine sufficient to confer immunity to pathogenicbacteria, viruses, or other microbes is determined by methods well knownto those skilled in the art in view of the guidance provided herein.This quantity will be determined based upon the characteristics of thevaccine recipient and the level of immunity required (as already notedabove). Where vaccines are administered by subcutaneous or intramuscularinjection, a range of 0.5 to 500 μg purified protein may be given. Asuseful in the present invention, such dosages are commonly sufficient toprovide about 1 μg, possibly 10 μg, even 50 μg, and as much as 100 μg,up to 500 μg of immunogenic protein, or immunogenic polypeptide, orimmunogenically active fragments thereof. In addition, more than onesuch active material may be present in the vaccine. Thus, more than oneantigenic structure may be used in formulating the vaccine, or vaccinecomposition to use in the methods disclosed herein. This may include twoor more individually immunogenic proteins or polypeptides, proteins orpolypeptides showing immunogenic activity only when in combination,either quantitatively equal in their respective concentrations orformulated to be present in some ratio, either definite or indefinite.

A vaccine composition for use in the processes disclosed herein mayinclude one or more immunogenic proteins, one or more immunogenicpolypeptides, and/or one or more immunogenically active immunogenscomprising antigenic fragments of said immunogenic proteins andpolypeptides, the latter fragments being present in any proportionsselected by the use of the present invention. The exact components, andtheir respective quantities, making up the vaccines, and vaccinecompositions, useful in the methods of the present invention aredetermined, inter alia, by the nature of the disease to be treated orprevented, the severity of such condition where it already exists, theage, sex, and general health of the recipient, as well the personal andprofessional experience and inclinations of the researcher and/orclinician utilizing these methods.

For a vaccine of the present invention, such as a sub-unit vaccine foruse against anthrax, specific non-limiting examples of this vaccinecomposition are those wherein said rPA is present at about 10 to 300μg/ml, preferably 50 to 300 μg/ml, or wherein said rPA is present atabout 100 to 300 μg/ml, more preferably about 150 to 250 μg/ml, and mostpreferably wherein said rPA is present at about 200 μg/ml.

In other examples, the dose of antigen, preferably rPA, to beadministered is at least about 5 μg, or at least about 10 μg, or atleast about 25 μg, or at least about 50 μg, or at least about 75 μg, orat least about 100 μg, or at least about 150 μg, or at least about 200μg, with a preferred dose of about 100 μg. A preferred dose volume isabout 0.5 ml.

The present invention further relates to a vaccine comprising a purifiedantigen, preferably an anthrax antigen, more preferably anthraxprotective antigen (PA) and most preferably a recombinant anthraxprotective antigen (rPA), such as that described in FIG. 2 of WO2007/122373, pub. 1 Nov. 2007, the disclosure of which is herebyincorporated by reference in its entirety, or that described inWO02/04646, bound to an adjuvant, preferably an alum-based adjuvant. Ina preferred embodiment, the present invention relates to a sub-unitvaccine comprising alum-bound rPA, at least about 2 mM and mostpreferably about 4 mM phosphate salt and at about pH 7.1.

A brief description of the rPA drug product manufacturing process ispresented below, along with a process diagram (FIG. 1). Drug substanceis removed from −70° C. storage and allowed to thaw at 2-8° C. While thebulk drug substance solution is thawing, three different solutions areprepared and utilized in the formulation process, namely Solutions A toC. Solution A is the stock 0.5 M phosphate buffer, which is subsequentlyused to produce Solution C (phosphate buffered 0.9% saline, containing0.04% Tween 20). It is Solution C that is used to dilute the rPA drugsubstance to the required concentration. Solution B comprises 1.22%saline, which is used to balance up the osmolality of the alhydrogel.The differences between the original low phosphate process and themodified high phosphate process is the concentration and pH of phosphatein Solution C.

Once the rPA drug substance is thawed and the rPA protein concentrationdetermined by the A280 nm in-process assay, this material was sterilefiltered along with the appropriate amounts of Solution C and SolutionB, into the formulation vessel. Once mixed, sterile alhydrogel wasadded, with continuous mixing to produce the drug product formulation,i.e. 200 ug/ml rPA, 0.26% alhydrogel in phosphate buffered 0.9% saline.The formulation is stirred for 2 hours before being filled into syringesor other appropriate containers.

It was found that for the low phosphate formulation, the pH at releasewas 5.9 for the resulting materials used in the clinic. In contrast thepH of the high phosphate material was approximately 7.2 at release.Consequently it can be concluded that the increase in phosphateconcentration was successful in its aim of controlling the pH at aneutral/physiological value.

A mouse challenge potency assay was used to test and release all batchesof drug product. This assay demonstrated that the Drug Products were allpotent when manufactured.

In order to be able to make comparative assessments of batch potency,the ED₅₀ value (a measure of effective dose) was derived from the assaydata. Surprisingly, this showed that the high phosphate batches produceda significant enhancement in the potency when compared with the lowphosphate batches. Moreover, the data from the high phosphateformulation were much more consistent than those from the low phosphateformulation (as indicated by the smaller error bars in FIG. 2). Thus,the modification to the phosphate concentration unexpectedly resulted ina marked enhancement of the drug product potency.

To determine the acceptable operating range for phosphate in the rPAdrug product process/formulation a series of studies have been performedto evaluate the effect of phosphate concentration upon both the proteinand the alum colloid. The results of these studies are presented below.

To evaluate the effect of phosphate on the alhydrogel particles the zetapotential of the alum colloidal particles was determined. Drug productand alum diluent were freshly formulated with a phosphate concentrationrange of 0.25 mM to 5 mM and the zeta potential was measured at T=day 0and T=day 7. The results revealed that with increasing phosphateconcentration the zeta potential became more negative, with the point ofno charge being between 2 and 3 mM phosphate (FIG. 3). It was alsoevident that the zeta potential, of both the drug product and thediluent, had not reach equilibrium during formulation but continued tochange over the preceding 7 days. This data indicated that thephosphate-alum interaction continued following formulation. However, byextrapolation to the zeta potential drug product batch data, whichindicated that the high phosphate drug product batches had a zetapotential of approximately −20 mV (FIG. 5), the interaction may bereaching completion by day 7.

Comparison of the rPA drug product and diluent zeta potential valuesindicated that at low phosphate levels (0.25 mM) the bound proteinmodified the zeta potential of the formulation, however this effect wasnot evident when the zeta potential became negative with increasingphosphate.

It is well established that phosphate concentration has a marked effectupon the ability of rPA to bind to alhydrogel. Early in drug productdevelopment, the effect of phosphate on rPA-alum interaction wasevaluated, however this was performed using a phosphate range of 10 to400 mM. This experiment has been repeated, except that this time theeffect of ≦10 mM was evaluated.

Increasing the concentration of phosphate inhibited rPA-alum binding(FIG. 4). At the 10 mM level, the % unbound rPA was at 12%, which waswithin the FDA guideline value of 30% unbound antigen.

Langmuir analysis was used to assess the effect of phosphate on rPAbinding to alhydrogel. This analysis is used to determine the adsorptivecoefficient (measure of binding strength) and adsorptive capacity(binding capacity) of molecules binding to particles.

As shown in FIG. 5, the adsorptive coefficient of rPA binding toalhydrogel was effected by the phosphate concentration, producing abiphasic curve. Following a steep drop in value, the adsorptivecoefficient reached a plateau around 3-4 mM phosphate, which suggestedthat phosphate ions were capable of modulating the strength of rPA-alumbinding. Certainly the effect of phosphate ions on the surface charge ofthe alum, making it more negative, would support this observation. TherPA protein is an acidic protein that would have a stronger interactionwith the positively charged alum particles as opposed to thephosphate-modulated negatively charged particles.

What is also evident from the Langmuir analysis is that the adsorptivecapacity of the alum also changed with increasing phosphateconcentration. This binding capacity parameter was starting to reach aplateau around 3-4 mM. It should be noted that this adsorptive capacitywas in excess of what was required to bind rPA at 200 ug/ml with aconcentration of 0.26% alhydrogel.

The specialized drug product far UV circular dichroism technique wasused to evaluate the effect of phosphate concentration on rPA secondarystructure in drug product. rPA Drug Product was formulated with a rangeof phosphate concentrations (0.25 mM to 10 mM) and the dichroism spectrawere determined between 190 nm and 250 nm. All the spectra (not shown)produced were characteristic of rPA with a minima at 208 nm and aplateau region around 216 nm (FIG. 6). Furthermore, all the spectraessentially overlapped, indicating that phosphate concentration was notaffecting the secondary structure of the rPA protein in freshlyformulated drug product.

Similarly, intrinsic fluorescence was used to evaluate the effect ofphosphate concentration on rPA tertiary structure in drug product. rPADrug Product was formulated with a range of phosphate concentrationsranging from 0.25 mM to 10 mM and the fluorescence emission spectra weredetermined. As shown in FIG. 7, all the spectra effectively overlappedeach other, irrespective of the phosphate concentration, indicating thatthere was no effect upon λmax and hence protein tertiary structure

Both intrinsic fluorescence and far UV circular dichroism indicated thatphosphate concentration did not effect the rPA secondary/tertiarystructure in freshly formulated drug substance. However, because it isknown that the rPA protein physically binds to the alum particles andbecomes immobilized, we determined whether there was a difference inrPA-alum binding, which did not significantly perturb rPA structure uponbinding, a thermal denaturation approach was adopted. DSC analysis wasused as an alternative measure of rPA-alum interaction. It was reasonedthat immobilizing rPA on alum would reduce heat capacity changesassociated with protein melting because of the restricted movement ofthe anchored protein and this would be detected by DSC.

When differential scanning calorimetry (DSC) was applied to freshlyformulated drug product over a phosphate concentration range of 0.25 mMto 50 mM, no melting transition was detected with the 0.25 mMformulation. As the phosphate concentration increased to 2 mM, atransition was detected, which increased in both enthalpy and in meltingtemperature with increased phosphate concentration (FIG. 8).

Overall, the DSC supported the observation that increasing phosphatelevels reduced the strength of binding of rPA to alum, as evident by theincreased heat capacity of the protein. The data also suggests that thestability of the protein increases inversely proportional to the amountit is bound to the alum, as shown be the increasing melting temperaturevalue. Although with the values for 10 and 50 mM phosphate the signalfrom unbound rPA would start to become significant and would contributedisproportionately to the isotherms.

The effectiveness of the thermal denaturation approach to evaluate therPA structure within drug product, as demonstrated in the DSC data,suggested that this could also be applied to intrinsic fluorescence.Hence, fluorescence intensity/thermal denaturation was used to determinethe effect of phosphate on the melting temperatures of the protein boundto alhydrogel (FIG. 9). Similar to that shown with DSC, drug productsformulated in low phosphate (0.25 mM to 2 mM) revealed little or noprotein melting transition in the 40-50° C. range, whereas a transitionwas detected at 3-10 mM, around 46° C. When the data is plotted asmelting temperature (midpoint of thermal transition) against phosphateit can be seen that the biphasic curve starts to reach a plateau at 3 mMphosphate.

In order to assess whether the increase in phosphate results in the rPAprotein binding in a different manner or orientation, studies wereperformed using the epitope recognition assay (also known as the DrugProduct immunoassay). This is effectively a protein structure assay thatmeasures the ability of selected monoclonal antibodies to recognizespecific rPA domain 4 epitopes. Domain 4 was chosen since there isevidence that this region of the protein is crucial for conferringantibody protection (Flick-Smith et al, Infect Immun., Vol. 70(3), pp.1653-6 (2002)), A recombinant carboxy-terminal domain of the protectiveantigen of Bacillus anthracis protects mice against anthrax infection).

Two monoclonal antibodies raised against separate rPA domain 4 epitopeswere used to evaluate rPA binding: (1) a stability-indicating epitope(C3 clone) and (2) a protective/stability-indicating epitope (2D4Jclone). Epitope mapping of the two antibodies demonstrated that theywere both binding to separate loop regions within rPA domain 4; moreoverthe 2D4J clone was specifically binding to the receptor binding region,which would account for its protective properties.

Epitope recognition analysis was performed with increasingconcentrations of phosphate. With both monoclonals there was a reductionin antibody binding as a result of the increase in phosphateconcentration (FIG. 10). With this assay it was not possible to evaluatea large number of phosphate concentrations due to limitations incapacity. However, from the limited number of phosphate concentrationsit was seen that the immunoreactivity with both monoclonals declinedrapidly up to an inflexion point around 2.5 mM, after which the rate ofdecline in immunoreactivity was decreased. This reduction inimmunoreactivity was not due to loss of protein since all rPAeffectively bound to the alum.

One unexpected conclusion of the above experiments was that phosphatemust henceforth be considered as a drug product formulation criticalparameter, due to its effect upon both the formulation characteristicsand potency. This is particularly important since up until this pointthe drug production process was not designed to manufacture rPA drugproduct with a fixed phosphate concentration. With this old process, rPAdrug substance was diluted with a phosphate buffer to reach a specificrPA protein concentration. Since the rPA concentration of the drugsubstance could potentially vary from the target concentration by afactor of about 17%, the amount of diluting phosphate buffer changeddepending on the starting concentration of the drug substance. Since itwas the volume of this phosphate that dictated the overall phosphateconcentration, the levels of this inorganic ion would varyappropriately. Subsequent modifications to the drug product process havebeen made to ensure that a constant phosphate concentration wasformulated, irrespective of the drug substance concentration at thestart of the process.

What is claimed is:
 1. An anthrax vaccine, consisting of atherapeutically effective amount of recombinant anthrax protectiveantigen (rPA) bound to aluminum hydroxide, and a phosphate salt at aconcentration in the range of 2.5 to 4.5 mM.
 2. The anthrax vaccine ofclaim 1, wherein said phosphate salt concentration is about 4 mM.
 3. Theanthrax vaccine of claim 1, wherein said rPA is present at about 100 to300 μg/ml.
 4. The anthrax vaccine of claim 1, wherein said rPA ispresent at 150 to 250 μg/ml.
 5. The anthrax vaccine of claim 1, whereinsaid rPA is present at 10 to 300 μg/ml.
 6. The anthrax vaccine of claim1, wherein the pH of said vaccine is in the range of 7.0 to 7.2.
 7. Theanthrax vaccine of claim 1, wherein said rPA is presented at about 100μg/ml, said phosphate is present at about 4.0 mM, said aluminumhydroxide is present at about 0.26% by weight and wherein the pH of saidvaccine is about 7.1.
 8. The anthrax vaccine of claim 1, wherein saidrPA is present at about 50 to 300 μg/ml.
 9. The anthrax vaccine of claim1, wherein said phosphate salt concentration is present in the range of3.8 mM to 4.2 mM.
 10. The anthrax vaccine of claim 1, wherein saidphosphate salt concentration is present in the range of 3.9 mM to 4.1mM.
 11. The anthrax vaccine of claim 1, wherein said phosphate saltconcentration is present in the range of 3.5 mM to 4.5 mM.
 12. A methodof protecting against a disease caused by an anthrax infectioncomprising administering to a mammal at risk of such an infection atherapeutically effective amount of the vaccine composition of claim 1.13. The method of claim 12, wherein said mammal is a human being.
 14. Amethod of protecting against a disease caused by an anthrax infectioncomprising administering to a mammal at risk of such an infection atherapeutically effective amount of the vaccine composition of claim 7.15. A method of treating a bacterial infection in a mammal, comprisingadministering to a mammal afflicted with such infection atherapeutically effective amount of the vaccine composition of claim 1.